Compounds and methods for modulating cell-adhesion mediated drug resistance

ABSTRACT

Peptides and methods of their use for inhibiting drug and radiation-therapy resistance in cancerous cells in which efficacy of chemotherapy and/or radiotherapy of a patient is enhanced by administration of an effective amount of a peptide that inhibits cell adhesion mediated drug resistance (CAM-DR). Preferably, the peptide comprises D-amino acids having the sequence: 
     
       
         kmviywkag  (RZ-3) 
       
     
     or is a variant or modified version thereof. The peptide is preferably administered to the patient prior to chemotherapy and/or radiation therapy. Inhibition of cell adhesion mediated drug resistance (CAM-DR) by RZ-3 in multiple myeloma cells is disclosed.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/795,484, filed Mar. 1, 2001, which claims the benefit of U.S.Provisional Application Ser. No. 60/186,198, filed Mar. 1, 2000.

GOVERNMENT SUPPORT

The subject matter if this application has been supported by a researchgrant from the National Institutes of Health under grant number R01CA77859. Accordingly, the government may have certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to the use of pharmaceutical agents tomodulate cell-adhesion. In particular, the invention relates to the useof peptides for inhibiting cell-adhesion and enhancing the efficacy ofthe chemotherapeutic and/or radiation treatments in the treatment ofcancer.

BACKGROUND OF THE INVENTION

Cell adhesion is a complex process that is important for maintainingtissue integrity and generating physical and permeability barrierswithin the body. Cell adhesion is mediated by specific cell surfaceadhesion molecules (CAMs). There are many different families of CAMs,including the immunoglobulin, integrin, selectin and cadherinsuperfamilies, and each cell type expresses a unique combination ofthese molecules.

Although cell adhesion is required for certain normal physiologicalfunctions including wound repair, there are situations in which celladhesion is undesirable. For example, many pathologies, such asmetastasis, autoimmune diseases, and inflammatory diseases, involveabnormal cellular adhesion. Cell adhesion may also play a role in graftrejection. In such pathologies, modulation of cell adhesion may bedesirable.

Multiple Myeloma and Treatment Resistance

Multiple myeloma is an incurable malignancy of the plasma cellcharacterized by migration and localization to the bone marrow wherecells then disseminate and facilitate the formation of bone lesions.Despite initial responses to chemotherapy, myeloma patients ultimatelydevelop drug resistance and become unresponsive to a wide spectrum ofanticancer agents, a phenomenon known as multidrug resistance (MDR). Thedevelopment of resistance to front-line chemotherapeutic drugs, such asmelphalan (an alkylating agent) and doxorubicin (an anthracycline), is amajor factor responsible for treatment failure; thus, the mediansurvival of 3.5 years has remained largely unchanged for the past threedecades.

There are many potential mechanisms for drug resistance in myelomaincluding reduction in intracellular drug accumulation due tooverexpression of MDR1/Pglycoprotein, alterations in drug targets suchas topoisomerase II, enhanced DNA repair, and overexpression ofanti-apoptotic proteins such as bcl-2. These mechanisms are chiefly dueto alterations in the malignant cell itself and can be studied using invitro human myeloma cell. However, these mechanisms alone cannot accountfor all drug resistance, nor are they likely to explain cell survivalfollowing initial cytotoxic drug exposure. Additional mechanismsconferring low level drug resistance are believed to play importantroles in the survival and expansion of the malignant cell population.Recent studies have demonstrated that certain resistance mechanisms areobserved only in vivo, suggesting that interactions between malignantcells and the surrounding microenvironment may be important indetermining response to chemotherapeutic drug. Factors that allow fortumor cell survival following initial drug exposure need to beidentified because these factors may eventually allow for expression ofgenes associated with acquired drug resistance. In addition, throughunderstanding the mechanism that suppresses drug-induced apoptosis aswell as other mediators of drug resistance, improved therapies can bedeveloped which interfere with, or inhibit the resistance.

It is known that intercellular interactions can contribute to tumor cellsurvival during exposure to cytotoxic stresses such as radiation. It isalso known that certain resistance mechanisms may only be functional invivo, where tumor cells continue to interact with environmental factorssuch as extracellular matrix (ECM) and cellular counter-receptors. Forexample, Teicher et al. showed that mammary tumors made resistant toalkylating agents in vivo are sensitized to cytotoxic drugs once removedfrom the animal, Tumor resistance to alkylating agents conferred bymechanisms operative only in vivo, Science 247:1457,1990. Adhesiveinteractions between same cell types are known to confer resistance toalkylating agents via alterations in cyclin dependent kinase inhibitorssuch as p27^(kipl), although the cell surface molecules mediating thistype of kinetic resistance have yet to be identified. In addition,adhesion to ECM has been reported to induce P-glycoprotein expressionand confer doxorubicin resistance in rat hepatocytes.

The integrin family of cellular adhesion molecules is a major class ofreceptors through which cells interact with extracellular matrixcomponents (ECM). Recent evidence has implicated the integrins as beingclosely involved in the pathology of many diseases. Integrins have beenshown to participate in intracellular signal transduction pathways thatmay contribute to tumor cell growth and survival.

Experimental evidence has implicated the PI integrins and fibronectin asplaying a part in apoptotic suppression and cell survival. For example,Zhang et al. has demonstrated that fibronectin adhesion through α₅β₁,(VLA-5) prevents cells from undergoing serum-starvation inducedapoptosis by upregulating Bcl-2. The alpha 5 beta I integrin supportssurvival of cells on fibronectin and up-regulates Bcl-2 expression, ProcNatl Acad Sci USA 92(13):6161, 1995.

Similar observations are made by Scott et al. and Rozzo et al., whofound that anti-β₁ antibodies and antisense oligonucleotides,respectively, enhanced chromatin condensation and nucleosomal DNAladdering, characteristics of cells committed to apoptosis. Fibronectinsuppresses apoptosis in normal human melanocytes through anintegrin-dependent mechanism, J Invest Dennaioi 108: 147, 1997.Induction of apoptosis in human neuroblastoma cells by abrogation ofintegrin mediated cell adhesion, Int J Cancer 70:688,-1997. PI integrinactivation through interactions with ECM components such as fibronectindirectly decreases DNA strand breaks in tumor derived endothelial cellsexposed to a number of DNA damaging agents, including etoposide andionizing radiation.

The α₄β₁ (Very Late Activation Antigen 4, or VLA-4), α₅β₁ (VLA-5), andα₄β₇ heterodimers are the major fibronectin receptors of the integrinfamily. Although VLA-5 and α₄β₇ expression are variable in most B cellsduring malignancy, VLA-4 is strongly expressed in myeloma cellscollected from bone marrow. VLA-4 is unique among the integrins as it isthe only heterodimer to have been shown to mediate cell-ECM as well ascell-cell interactions. VLA-4 binds to the CS-1 region of fibronectin aswell as to vascular cell adhesion molecule-1 (VCAM-1) via separatebinding sites. Adhesion to fibronectin via VLA-4 has been shown toprolong eosinophil survival and to downregulate FAS antigen expression,leading to a decrease in cell death. In early hematopoietic and germinalcenter B cells, adhesion to fibronectin or VCAM-1 via VLA-4 suppressesthe apoptotic pathway and contributes to positive selection.

As myeloma cells adhere in the bone marrow, they stimulate their owngrowth and cause osteoclast formation through the increased synthesisand secretion of cytokines such as IL-1β, TNF-β, M-CSF, and IL-6. IL6, apotent growth factor for myeloma cells, is secreted from both tumor andstromal cells in response to co-adhesion and VLA-4 ligation. VLA-4associates with or causes the phosphorylation f a number of signaltransduction molecules, including CD19 receptor-associated proteintyrosine kinases and focal adhesion kinase (pp125^(FAK), or FAK), whichis an upstream activator of mitogen activated protein kinase (MAPK),among other proteins. FAK plays a major role in suppressing apoptosisboth in adherent and suspension cells, and its cleavage by caspasesearly in the apoptotic process further emphasizes its importance withinthe cell.

Peptide and Cell-Adhesion

Peptides capable of modulating cell adhesion have been reported. U.S.Pat. No. 6,169,071 to Blaschuk , et al. discloses cyclic peptidescomprising a cadherin cell adhesion recognition sequence HAV and methodsfor modulating cadherin-mediated cell adhesion in a variety of contexts.U.S. Pat. No. 6,020,460 to Pierschbacher, et al. disclosedconformationally stabilized synthetic Arg-Gly-Asp-containing peptideswhich have increased affinity and selectivity for the vitronectinreceptor over that of linear, Arg-Gly-Asp-containing synthetic peptides.Such peptides are used to modulate cell-adhesion.

However, none of the above cited art references discloses or evensuggests the administration of peptides to enhance chemo- orradiotherapy, nor in situations when such drugs may help to overcomecell adhesion inhibition-mediated drug resistance. In contrast, andteaching away from the instant application, the rise of drug resistanttumor cells is usually associated with reduced migratory and invasiveability and a lower adhesion capacity (see abstract by Scotlandi K. etal., Multidrug resistance and malignancy in human osteosarcoma, CancerRes 1996 May 15;56(10):2434-9).

Accordingly, there is a need in the art for compounds that modulate celladhesion to improve, for example, the efficacy of chemotherapy andradiation therapy of cancer cells such as multiple myeloma without theaforementioned disadvantages. The present invention fulfills this needand further provides other related advantages that will become apparentto one of ordinary skill of the art upon reading the followingdisclosure.

BRIEF SUMMARY OF THE INVENTION

It is therefore an aspect of the present invention to provide animproved protocol for the treatment of cancer.

It is a further aspect of the present invention to provide a protocolcomprising the administration of pharmaceutical agents that disruptcancer cell adhesion in vivo to enhance the efficacy of chemotherapeuticand/or radiation treatments.

It is a further aspect of the present invention to provide a protocolcomprising the administration of peptides for inhibiting adhesion andthereby enhancing the efficacy of chemotherapeutic and/or radiationtreatments in the treatment of cancer.

It is yet a further aspect of the present invention to provide a methodof treating cancer that inhibits cell adhesion mediated drug resistance.

It is a further aspect of the present invention to provide a method thatenhances the efficacy of cytotoxic drugs and/or radiation in thetreatment of cancer.

In another embodiment, it is an aspect of the present invention toprovide factors which confer cell adhesion mediated drug resistance,wherein such factors provide novel and specific targets for thedevelopment of therapies that can interfere with or inhibit celladhesion mediated drug resistance.

It is a further aspect of the present invention to provide a rationalconnection between expression and function of the major integrinfibronectin receptors and the response to cytotoxic drugs in the humanmyeloma derived cell line RPMI 8226.

It is yet a further aspect of the present invention to provide changesin integrin expression and function in drug-resistant variants followingchronic drug exposure that permit assessment of the effects offibronectin on acute drug response in the drug sensitive parent line.

These and other aspects of the present invention will become obvious tothose skilled in the art upon review of the following disclosure.

Adhesion of cancer cells to fibronectin via integrins or fibronectininterferes with drug and/or radiation induced apoptosis. Disclosedherein are specific agents that can interfere with cell adhesionmediated drug resistance and thereby enhance the ability of cytotoxicdrugs and radiation to kill cancer cells. In particular, peptides aredisclosed as a class of drugs that can block cell adhesion and preventcell adhesion mediated drug resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates 8226/S myeloma cells adhered to fibronectin (FN)having a survival advantage over non-adhered cells following acutedoxorubicin exposure.

FIG. 2 illustrates Annexin V stained FN-adhered myeloma cells having alower apoptotic fraction compared to non-adhered cells following acutedrug exposure.

FIG. 3 illustrates the fact that RNA levels of Bcl-2 family members areunchanged following FN adhesion.

FIG. 4 illustrates the fact that intracellular doxorubicin concentrationis to unaffected by culturing cells on plastic, or FN.

FIG. 5 illustrates phenotypic analysis of 8226 cell surface FN receptorexpression by flow cytometry.

FIG. 6A illustrates drug resistance associated with α₄ expression inmelphalan resistant (8226/LR5) and revertant (LR5ood) cell lines

FIG. 6B illustrates drug resistance associated with α₄ expression indoxorubicin resistant (8226/DOX6) and revertant (DOX6ood) cell lines.

FIG. 7 illustrates the contribution of α₄ and α₅ integrin subunits to FNadhesion.

FIG. 8 illustrates K562 cells adhered to FN having a survival advantageover non-adhered cells following exposure to mitoxantrone and melphalan.

FIG. 9 illustrates the fact that K562 cells are resistant tomelphalan-induced apoptosis only following FN-specific adhesion.

FIG. 10 illustrates the fact that K562/VLA-4 overexpresses the α4integrin subunit.

FIG. 11 illustrates the fact that K562/pcDNA3.1 adheres to FN via VLA-5while K562/VLA-4 adheres to FN via VLA-4 and VLA-5.

FIG. 12 illustrates the fact that α4 and α5-mediated FN adhesion inducecell adhesion mediated drug resistance but the effects of each receptorare not additive.

FIG. 13 illustrates the fact that K562 cells adhered to FN are resistantto the cytotoxic effects of the BCR/ABL inhibitor AG957.

FIG. 14 illustrates the fact that phosphorylation of an 80 kDa proteinis detectable FN adhered K562 cells.

FIG. 15 illustrates the fact that integrin activation does notreconstitute BCR/ABL-associated tyrosine kinase activity in AG957treated cells.

FIG. 16 illustrates FN adhered cells maintaining the CAM-DR phenotypeduring AG957 exposure.

FIG. 17 illustrates resistance to cytotoxic drugs resulting fromadhesion of myeloma cells to fibronectin.

FIG. 18 illustrates upregulation of expression of VLA-4 and increasedadhesion to fibronectin due to selection for drug resistance tomelphalan and doxorubicin in myeloma cell lines.

FIG. 19 illustrates overexpression of VLA-4 by melphalan resistant anddoxorubin resistant cell lines.

FIG. 20 illustrates the fact that myeloma cells that express VLA-4 andVLA-5 undergo G1 arrest when adhered to fibronectin.

FIG. 21 illustrates thymidine incorporation.

FIG. 22 illustrates the percentage of cells in G1.

FIG. 23 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of 8226 cells.

FIG. 24 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of 8226 cells.

FIG. 25 illustrates the effect of (he D-amino acid peptide RZ-3 onadhesion of 8226 cells.

FIG. 26 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of U937 cells.

FIG. 27 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of 8226 cells, wherein the cells are treated with RZ-3 forthirty minutes and then adhered for two hours.

FIG. 28 illustrates two different forms of tumor-microenvironmentinteractions influencing drug response in cancer.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates 8226/S myeloma cells adhered to FN having a survivaladvantage over non-adhered cells following acute doxorubicin exposure(a) but not following melphalan exposure (b) in cell growth basedcytotoxicity assays. FN-adhered cells (---) are bound to FN-coatedplates 24 hours prior to one hour drug exposure and control cells aregrown in suspension (—). Response to doxorubicin is 12.6 fold lower inFN-adhered cells compared to non-adhered controls (IC50 values foradhered and nonadhered cells are of 4.85×10⁻⁷ M and 8.5×10⁻⁸ M,respectively). Data points are presented as cell viability determined byMTT cytotoxicity assay compared to untreated controls. Graphs arerepresentative experiments that are repeated 3 times in replicates of 4.

FIG. 2 illustrates Annexin V stained FN-adhered myeloma cells having alower apoptotic fraction compared to non-adhered cells following acutedrug exposure. 8226/S myeloma cells are exposed to 1 uM doxorubicin forone hour (a) or 50 uM melphalan for 24 hours (b), stained by Annexin V24 hours later, then analyzed by flow cytometry. Histograms are adjustedfor background staining in untreated cells, bars are the s.d. of threedifferent experiments; *, P<0.05.

FIG. 3 illustrates the fact that RNA levels of Bcl-2 family members areunchanged following FN adhesion. Drug sensitive-8226/S cells are adheredto FN-coated plates or grown in suspension for 24 hours after whichtotal RNA is collected and analyzed by RNase protection. Expressionlevels are normalized to the housekeeping genes GAPDH and L32.

FIG. 4 illustrates the fact that intracellular doxorubicin concentrationis unaffected by culturing cells on plastic, BSA, or FN. Following a 24hour incubation on each surface, 10 uM doxorubicin is added to each wellfor one hour and cells are analyzed for drug accumulation differences byflow cytometry. Bars are the s.d. of n=6 from two independentexperiments.

FIG. 5 illustrates phenotypic analysis of 8226 cell surface FN receptorexpression by flow cytometry. Integrin subunit expression by drugsensitive (8226/S), melphalan resistant (8226/LR5), and doxorubicinresistant (8226/DOX6) cell lines are analyzed using monoclonalantibodies for α₄, α₅, β₁ and β₇. Cells are incubated with anintegrin-specific mAb (—) or with irrelevent control Ab (---), followedby incubation with FITC-conjugated secondary Ab. 10,000 events areanalyzed for each sample using a FACScan machine (Becton-Dickinson),histograms are representative of three different experiments.

FIG. 6A illustrates drug resistance associated with α₄ expression inmelphalan resistant (8226/LR5) and revertant (LR5ood) cell lines.8226/LR5 are maintained in 5×10⁻⁵ M melphalan (LPAM) and LR5ood aremaintained out of drug for 20 weeks. α₄ expression is measured by flowcytometry and drug resistance is measured by MTT cytotoxicity analysis.Resistance values are reported as the IC50 dose of LPAM relative to8226/S. α₄ expression levels and melphalan resistance levels of 8226/LR5are found to be higher than 8226/S (P<0.05). α₄ expression and melphalanresistance of LR5ood are found to be equal to those of the 8226/S parentline. Bars are the s.d. of three different experiments.

FIG. 6B illustrates drug resistance associated with α₄ expression indoxorubicin resistant (8226/DOX6) and revertant (DOX6ood) cell lines.8226/DOX6 are maintained 6×10⁻⁸ M doxorubicin and DOX6ood are maintainedout of drug for 20 weeks. α₄ expression is measured by flow cytometryand drug resistance is measured by MTT cytotoxicity analysis. Resistancevalues are reported as the IC50 dose of doxorubicin relative to 8226/S.α₄ expression levels and doxorubicin resistance levels of 8226/DOX6 arefound to be higher than 8226/S (P<0.05). α₄ expression and doxorubicinresistance of DOX6ood are found to be equal to those of the 8226/Sparent line. Bars are the s.d. of three different experiments.

FIG. 7 illustrates the contribution of α₄ and α₅ integrin subunits to FNadhesion. Drug sensitive (8226/S), melphalan resistant (8226/LR5), anddoxorubicin resistant (8226/DOX6) are adhered to FN-coated wells(horizontal striped bars) for one hour. To determine percentage bindingdue to α₄ and α₅, some cells are pre-incubated with α₄ function blockingAb P4G9 (hatched bars) or α₅ function blocking Ab P1D6 (vertical stripedbars) for 15 minutes prior to application to wells. FN adhesion by8226/S is found to be mediated equally by α₄ and α₅ while FN adhesionfor both drug resistant cell lines is mediated only by α₄ (P<0.05), asdetermined by complete inhibition of adherence using the (α₄ blockingAb. Total FN adhesion mediated by α₄ is also higher in drug resistantlines compared to drug sensitive 8226/S (P<0.05). Values shown are the %of total cells applied to each well corrected for non-specific adhesionto BSA-coated wells. Bars are the s.d. of n=6 from three differentexperiments.

FIG. 8 illustrates K562 cells adhered to FN having a survival advantageover non-adhered cells following exposure to mitoxantrone and melphalan.Cell growth based MTT cytotoxicity assays are used to determine responseto (A) mitoxantrone and (B) melphalan (LPAM). FN adhered cells (---) arebound to FN coated plates 24 hours prior to 96 hour drug exposure andcontrol cells are grown in suspension (—). Data points are presented ascell viability determined by MTT dye reduction compared to untreatedcontrols. Graphs are representative experiments that are repeated 3times in replicates of 4.

FIG. 9 illustrates the fact that K562 cells are resistant tomelphalan-induced apoptosis only following FN-specific adhesion. Cellsare seeded on each matrix for two hours, exposed to 100 μM LPAM for 90minutes, washed, and incubated for 24 hours. Percent apoptosis isdetermined by Annexin V staining and flow cytometry, during which 5000events are analyzed for each sample. Bars represent standard deviationsand * denotes significant reduction in apoptosis by Student's t-test(p<0.05).

FIG. 10 illustrates the fact that K562/VLA-4 overexpresses the α4integrin subunit. K562/VLA-4, but not K562/pcDNA3.1, expresses the α4subunit protein on its surface, as determined by flow cytometry (A). α4mRNA is only present in K562/VLA-4 by RT-PCR analysis (B).

FIG. 11 illustrates the fact that K562/pcDNA3.1 adheres to FN via VLA-5while K562/VLA-4 adheres to FN via VLA-4 and VLA-5. Cell adhesion isdetermined using integrin blocking antibodies and a calorimetric assay.Cells are pretreated with β1 activating mAb B3B11 (1:100) and blockingmAb prior to application to FN coated wells for one hour. Absorbancevalues shown are the mean of three replicates, with background bindingto BSA subtracted from each. Experiments are repeated 3 times.

FIG. 12 illustrates the fact that α4 and α5-mediated FN adhesion inducecell adhesion mediated drug resistance but the effects of each receptorare not additive. Cells are pretreated with mAb B3B11, then with anti-α4blocking mAb (P4G9), anti-α5 blocking mAb (P1D6), or IgG3 isotypecontrol Ab prior to adhesion to FN coated dishes. Cells are exposed to100 μM LPAM for 90 minutes, washed, incubated for 24 hours, and analyzedusing Annexin V staining and flow cytometry. Bars are the standarddeviations of three independent experiments performed in duplicate, *denotes p<0.05 by Student's t-test.

FIG. 13 illustrates the fact that K562 cells adhered to FN are resistantto the cytotoxic effects of the BCR/ABL inhibitor AG957. The MTT assayis used to evaluate sensitivity of suspension grown and FN adhered K562cells treated with AG957 for 96 hours. The mean IC50 for suspensiongrown cells (—) is 3.60×10⁻⁶M compared to 12.95×10⁻⁶M for FN adheredcells (---). Graph shown is representative of three independentexperiments which are significantly different at p<0.05 by Student'st-test.

FIG. 14 illustrates the fact that phosphorylation of an 80 kDa proteinis detectable FN adhered K562 cells. Cells are adhered to FN for thetimes indicated or are kept in suspension, then analyzed for thepresence of phosphorylated tyrosine residues. Western blotting usinganti-phosphotyrosine mAb is used to determine activity and β-actin isused as a control for equal protein loading.

FIG. 15 illustrates the fact that integrin activation does notreconstitute BCR/ABL-associated tyrosine kinase activity in AG957treated cells. K562 cells are adhered to FN or kept in suspension for 24hours, after which they are exposed to 20 μM AG957 or DMSO (vehiclecontrol) for four hours. Proteins are separated by 10% SDS-PAGE andsubjected to anti-phosphotyrosine immunoblotting. AG957 abrogates allphosphotyrosine activity with the exception of the 80 kda protein, whichpersists to a small degree. Blot shown is representative of threeindependent experiments.

FIG. 16 illustrates FN adhered cells maintaining the CAM-DR phenotypeduring AG957 exposure. K562 cells are adhered to FN (striped bars) orkept in suspension (solid bars) for three hours, treated with AG957 (orDMSO vehicle control) for two hours, then exposed to 100 μM LPAM (orAcid-OH vehicle control) for 90 minutes. Apoptosis is analyzed 24 hourslater by Annexin V staining and flow cytometry. Graph is representativeof three independent experiments performed in duplicate. * indicates %apoptosis (FN vs. suspension) is significantly different (p<0.05 byStudent's t-test).

FIG. 17 illustrates resistance to cytotoxic drugs resulting fromadhesion of myeloma cells to fibronectin. Adhesion of myeloma cells toFN results in resistance to cytotoxic drugs. 8226 myeloma cells grown onFN-coated plates are less sensitive to the cytotoxic effects ofdoxorubicin and melphalan as determined by MTT cell viability assays andAnnexin V analysis.

FIG. 18 illustrates upregulation of expression of VLA-4 and increasedadhesion to fibronectin due to selection for drug resistance tomelphalan and doxorubicin in myeloma cell lines. Selection for drugresistance to melphalan and doxorubicin in myeloma cell lines results inupregulation of expression of VLA-4 and increased adhesion to FN. Humanmyeloma cell lines 8226 and U266 adhere to fibronectin (FN) through β1integrin interactions (VLA-4 and VLA-5). Melphalan and doxorubicinresistant variants of these cell lines show increased levels of VLA-4mediated adhesion to FN as determined by anti-integrin mAbs.

FIG. 19 illustrates overexpression of VLA-4 by melphalan resistant anddoxorubin resistant cell lines. Melphalan resistant (8226/LR5) anddoxor-ubicin resistant (8226/Dox6) cell lines overexpress VLA-4 (a4p Iintegin heterodimer), as determined by FACS analysis.

FIG. 20 illustrates the fact that myeloma cells that express VLA-4 andVLA-5 undergo G1 arrest when adhered to fibronectin. 96 well plates arecoated with 50 μg/ml fibronectin (FN), 0.01 percent BSA or two percentPoly-Hema (prevents adhesion to plastic) per well. Cells are allowed toadhere for one hour to fibronectin in serum free media, unadhered cellsare then washed and 200 μl RPNH media containing 5 percent FBS is addedback to each well. Growth is measured by MTT at 24, 48, 72 and 96 hours.

FIG. 21 illustrates thymidine incorporation. 96 well plates are coatedwith 50 μg/ml fibronectin (FN), 0.01 percent BSA or two percentPoly-Hema (prevents adhesion to plastic) per well. Cells are allowed toadhere for one hour to fibronectin in serum free media and, unadheredcells are removed and 200 μl RPMI media containing 5 percent FBS isadded back to each well. Cells are pulsed with two μCi 3H-thymidine forthree hours at each respective time point. A cell harvester is used tocollect DNA, and labeled DNA is counted on a scintillation counter.

FIG. 22 illustrates the percentage of cells in G1. Cells are allowed toadhere to fibronectin or poly-lysine coated plates for 48 or 76 hours.Unattached cells are removed by washing the plate with PBS beforeremoving adhered cells. Cells which adhere to fibronectin are removedwith PBS containing two mM EDTA. Cells are fixed, RNA is digested withRNase and the nuclei are stained with propidium iodide. Cell cycle isanalyzed by FACS and a Cell Lysis software program is used to determinethe percentage of cells in G1.

Overexpression of CDK inhibitors correlates with a decrease insensitivity to chemotheurapeutic agents. Cells that have been arrestedin G1 have an increased capacity to repair DNA damage induced by DNAdamaging agents such as cis-platinum and doxorubicin. Thus,physiological mediators of G1 arrest are important targets forsensitizing cells to existing chemotheurapeutic agents

FIG. 23 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of 8226 cells. 8226 myeloma cells are treated with varyingconcentrations of RZ-3, (ranging from 20 μg/ml to 100 μg/ml) followed byplating on to BSA or fibronectin (FN) coated plates. Cells do not adheresignificantly to BSA but do significantly adhere to FN. RZ-3 iseffective in preventing FN adhesion.

FIG. 24 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of 8226 cells. 8226 myeloma cells are treated with varyingconcentrations of RZ-3, (ranging from 1.25 μg/ml to 20 μg/ml) followedby plating on to BSA or fibronectin (FN) coated plates. Cells do notadhere significantly to BSA but do significantly adhere to FN. RZ-3 iseffective in preventing FN adhesion.

FIG. 25 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of 8226 cells. 8226 myeloma cells are treated with varyingconcentrations of RZ-3, (ranging from 1.25 μg/ml to 20 μg/ml) followedby plating on to BSA or fibronectin (FN) coated plates. Cells do notadhere significantly to BSA but do significantly adhere to FN. RZ-3 iseffective in preventing FN adhesion.

FIG. 26 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of U937 cells. U937 cells are treated with varyingconcentrations of RZ-3, (ranging from 5 μg/ml to 80 μg/ml) followed byplating on to BSA or fibronectin (FN) coated plates. Cells do not adheresignificantly to BSA but do significantly adhere to FN. RZ-3 iseffective in preventing FN adhesion.

FIG. 27 illustrates the effect of the D-amino acid peptide RZ-3 onadhesion of U937 cells. 8226 myeloma cells are treated with varyingconcentrations of RZ-3, (ranging from 1.25 μg/ml to 20 μg/ml) for 30minutes, followed by plating on to BSA or fibronectin (FN) coatedplates. Cells do not adhere significantly to BSA but do significantlyadhere to FN. RZ-3 is effective in preventing FN adhesion.

FIG. 28 illustrates the soluble form of tumor-microenvironmentinteraction (IL-6) as well as the direct contact form oftumor-microenvironment interaction (ECM).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an upstream primer.

SEQ ID NO:2 is a downstream primer.

SEQ ID NO:3 is an upstream primer.

SEQ ID NO:4 is a downstream primer.

SEQ ID NO:5 is the amino acid sequenceLys-Met-Val-Ile-Tyr-Trp-Lys-Ala-Gly.

SEQ ID NO:6 is the RZ-3 peptide, the amino acid sequenceLys-Met-Val-Ile-Tyr-Trp-Lys-Ala-Gly, wherein each amino acid is aD-amino acid.

DETAILED DESCRIPTION OF THE INVENTION

Peptides as described herein may comprise residues of L-amino acids,D-amino acids, or any combination thereof. Amino acids may be fromnatural or non-natural sources, provided that at least one amino groupand at least one carboxyl group are present in the molecule. The 20L-amino acids commonly found in proteins are identified herein by theconventional one-letter abbreviations known in the art, and thecorresponding D-amino acids are designated by a lower case one lettersymbol. Peptides may also contain one or more rare amino acids (such as4-hydroxyproline or hydroxylysine), organic acids or amides and/orderivatives of common amino acids, such as amino acids having theC-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester)or amidated and/or having modifications of the N-terminal amino group(e.g., acetylation or alkoxycarbonylamino), with or without any of awide variety of side chain modifications and/or substitutions (e.g.,methylation, benzylation, t-butylation, tosylation, alkoxycarbonylamino,and the liked). Such modifications and derivatives of a peptidesequence, and others known to those of skill in the art, are hereintermed “variants.” Preferred derivatives include amino acids having anN-acetyl group (such that the amino group that represents the N-terminusof the linear peptide is acetylated) and/or a C-terminal amide group(i.e., the carboxy terminus of the linear peptide is amidated). Residuesother than common amino acids that may be present include, but are notlimited to, penicillamine, tetramethylene cysteine, pentamethylenecysteine, mercaptopropionic acid, pentamethylene-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, aminoadipic acid, m-aminomethylbenzoic acid anddiaminopropionic acid.

Peptides as described herein may be synthesized by methods well known inthe art, including recombinant DNA methods and chemical synthesis.Chemical synthesis may generally be performed using standard solutionphase or solid phase peptide synthesis techniques, in which a peptidelinkage occurs through the direct condensation of the amino group of oneamino acid with the carboxy group of the other amino acid with theelimination of a water molecule. Peptide bond synthesis by directcondensation, as formulated above, requires suppression of the reactivecharacter of the amino group of the first and of the carboxyl group ofthe second amino acid. The masking substituents must permit their readyremoval, without inducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (see Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy,which are now well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

The peptides disclosed here in may be modified by attachment of a secondmolecule that confers a beneficial property upon the peptide, such asincreased half-life in the body, for example pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

Covalent attachment of a molecule or solid support may generally beachieved by first reacting the support material with a bifunctionalreagent that will also react with a functional group, such as ahydroxyl, thiol, carboxyl, ketone or amino group, on the modulatingagent. A preferred method of generating a linkage is via amino groupsusing glutaraldehyde. A modulating agent may be linked to cellulose viaester linkages. Similarly, amide linkages may be suitable for linkage toother molecules such as keyhole limpet hemocyanin or other supportmaterials.

Although peptides as described herein may preferentially bind tospecific tissues or cells, and thus may be sufficient to target adesired site in vivo, it may be beneficial for certain applications toinclude an additional targeting agent. Accordingly, a targeting agentmay also, or alternatively, be linked to a modulating agent tofacilitate targeting to one or more specific tissues. As used herein, a“targeting agent,” may be any substance (such as a compound or cell)that, when linked to a modulating agent enhances the transport of themodulating agent to a target tissue, thereby increasing the localconcentration of the modulating agent. Targeting agents includeantibodies or fragments thereof, receptors, ligands and other moleculesthat bind to cells of, or in the vicinity of, the target tissue. Knowntargeting agents include serum hormones, antibodies against cell surfaceantigens, lectins, adhesion molecules, tumor cell surface bindingligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes andthose drugs and proteins that bind to a desired target site.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds. Theuse of certain specific drugs within the context of the presentinvention is discussed below.

Within certain aspects of the present invention, one or more modulatingagents as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulating agents in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. A peptide may, but need not, beencapsulated within liposomes using well known technology. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration. For certain topical applications,formulation as a cream or lotion, using well known components, ispreferred.

Integrin-mediated adhesion influences cell survival and can preventprogrammed cell death. Drug-sensitive 8226 human myeloma cells expressboth VLA-4 (α₄β₁) and VLA-5 (α₅β₁) integrin fibronectin (FN) receptors,and are relatively resistant to the apoptotic effects of doxorubicin andmelphalan when pre-adhered to FN, as compared with cells grown insuspension. This cell adhesion mediated drug resistance, or CAM-DR, isnot due to reduced drug accumulation or upregulation of anti-apoptoticBcl-2 family members. As determined by flow cytometry, myeloma celllines selected for drug resistance, with either doxorubicin ormelphalan, overexpress VLA-4. Functional assays reveal a significantincrease in α₄-mediated cell adhesion in both drug-resistant variantscompared with the drug-sensitive parent line. When removed fromselection pressure, drug-resistant cell lines revert to a drug sensitiveand α₄-low phenotype.

It is therefore disclosed herein that FN-mediated adhesion confers asurvival advantage for myeloma cells acutely exposed to cytotoxic drugsor radiotherapy by inhibiting drug-induced apoptosis. This findingexplains how some cells survive initial drug exposure or radiotherapyand eventually express classical mechanisms of drug resistance such asMDR1 overexpression.

EXAMPLE ONE

Materials and Methods

Cell and culture condition

The RPMI 8226 human myeloma cell line (8226/S) is obtained from theAmerican Type Culture Collection (Rockville, Md.). The drug resistantcell lines, 8226/DOX6 and 8226/LR5, are selected from 8226/S usingstep-wise increases of doxorubicin and melphalan, respectively, asdescribed for example by Dalton W. et al., Characterization of a newdrug-resistant human myeloma cell line that expresses P-glycoprotein,Cancer Res 46:2125, 1986 and Bellamy W T, et al., Development andcharacterization of a melphalan resistant human multiple myeloma cellline, Cancer Res 51:995, 1991. All cells are grown in suspension in RPMI1640 medium supplemented with 5 percent fetal bovine-serum, 1 percent(vol/vol) penicillin (100 U/mL), streptomycin (100 U/mL), and 1 percent(vol/vol) L-glutamine (all from GIBCO-BRL, Grand Island, N.Y.). Cellsare maintained at 37° C. in 5 percent CO₂-95 percent air atmosphere andsubcultured every 6 days.

Drugs

Melphalan (L-phenylalanine nitrogen mustard, LPAM) is obtained fromSigma-Aldrich (St Louis, Mo.) and dissolved in acidified ethanol.Doxorubicin is obtained from Sigma Aldrich and dissolved in sterileddH₂O.

Cytotoxicity assays

96-Well immunosorp plates (Nunc, Denmark) are coated with 50 μL (40μg/mL) FN (GIBCO) or bovine serum albumin (BSA) overnight and onepercent BSA is used to block nonspecific binding sites in the wells forone hour before the is experiment. Wells are washed with serum-free RPMI1640 and aspirated. 8226/S cells are washed once and resuspended inserum-free RPMI 1640; then 4×10⁴ cells per well (FN coated) or 8×10³cells per well (BSA coated) are added to each plate. Cells are incubatedfor one hour at 37° C. and five percent CO₂, washed with serum freemedia twice, and put back into serum-containing media. Following 24hours in a tissue culture incubator, 20 μL of diluted drug or vehiclecontrol is added to each well for one hour, after which media is removedand replaced by drug-free media. Following a 96-hour incubation, 50 μLMTT dye (Sigma) is added to each well for four hours. Plates are thencentrifuged and each well aspirated. Dye is solubilized with 100 μL DMSOand plates are read at 540 nm on an automated microtiter plate reader. Ablank well containing only media and drug is also run as a control inall experiments. IC₅₀ values are calculated by linear regression ofpercent survival versus drug concentration.

Annexin V apoptotic analysis

Cells, 1×10⁶, are attached to FN-coated 6-well plates (Biosource,Camarillo, Calif.) for one hour in serum-free media and nonadhered cellsare removed with two washes. Fresh media with serum is added to theplates, which are incubated for 24 hours; 1×10⁶ cells are also added touncoated 6-well plates (Boeringer-Mannheim, Indianapolis, Ind.). Cellsare exposed to 1 μmol/L doxorubcin for one hour (plus a 24-hourdrug-free incubation period) or 50 μmol/L LPAM for 24 hours. Cells arethen collected with 5 mmol/L EDTA/PBS and washed. Phycoerythrin (PE)- orfluorescein isothiocyanate (FITC)-conjugated Annexin V (Clontech, PaloAlto, Calif.) is then added to 1×10⁵ cells and 5,000 to 10,000 eventsare analyzed on a FACScan machine (Becton Dickinson).

RNA collection and cDNA synthesis from FN-adhered myeloma cells

Cells, 1×10⁶, are attached to FN coated 6-well plates as previouslydescribed. Total cellular RNA is collected on 3 separate days usingTRIzol reagent (GIBCO). RNA is quantitated on a spectophotometer at 260nm and one μg is DNAse treated and requantitated. A single large scalecDNA reaction is prepared for each sample for use in PCR reactions. A 40μL reverse transcription reaction containing 200 ng RNA, 1×PCR buffer(10 mmol/L Tris, pH 8.3-50 mmol/L MKCL-1.5 mmol/L MgCl₂), 1 mmol/Lconcentrations each of dATP, dGTP, dCTP, and dTTP; 200 pmol randomhexamers, 40 U RNAse inhibitor, and 12 U avian megalovirus reversetranscriptase (Boeringer-Mannheim) is prepared on ice then incubated at42° C. for one hour, 99° C. for 10 minutes, and quick chilled to 4° C.

RNase protection assay

Twenty micrograms of RNA is isolated from 8226/S cells grown insuspension or adhered to FN using TRIzol reagent and resuspended inhybridization buffer. Bcl-2 family specific probes are synthesized usinga template set from Riboquant (San Diego, Calif.) and labeled using[α-³²p]UTP and T7 polymerase. Probes are then column purified,quantitated on a scintillation counter, and 5×10⁵ cmp is added to eachsample. The hybridization reaction is carried out overnight at 56° C.Samples are then RNase treated for 45 minutes at 30° C., hybridizedprobes are extracted with chloroform: isoamyl alcohol and precipitatedusing 100 percent ethanol. Samples are then electrophoresed on a fivepercent polyacrylamide gel (7 mmol/L Urea), dried down, exposed to film,and analyzed by densitometry software (ImageQuaNT, Molecular Dynamics,Inc, Sunnyvale, Calif.). Unhybridized probes are used as size standardsfor each gene analyzed. Expression of Bcl-2, Bcl-Xl, Bcl-Xs, BAX, Bik,Bad, Bcl-w, Bak, Mcl-1, and Bfl I are quantitated by normalizing toGAPDH and L32 expression.

RT-PCR Analysis of BCL-2 Family Gene and Drug Transporter Expression

BCL-2 amplification is performed essentially as described by Tu Y etal., Upregulated expression of Bcl-2 in multiple myeloma cells inducedby exposure to doxorubicin, etoposide, and hydrogen peroxide, Blood88:1805,1996. Briefly, 20 μL of PCR reaction mixture (1×PCR buffer, 50pmol of BCL-2 specific amp limers, 0.25 U Taq polymerase[Boehrin.-erMannheim], 1.25 μCi [α-³²p]-dCTP) is added to 5 μL cDNA,followed by incubation at 94° C. for 5 minutes and then 26 cycles of 94°C. for one minute, 72° C. for one minute, and a final extension at 72°C. for five minutes in a thermal cycler (Perkin-Elmer Cetus). Histone3.3 is amplified as described by Futscher Bwet al., Quantitativepolymerase chain reaction analysis of MDR1 mRNA in multiple myetoma celllines and clinical specimens, Anal Biochem 213:414, 1993, and is used asa control for RNA integrity and quantity. Bcl-XI and -Xs are amplifiedessentially as described by Benito A, et al., Apoptosis induced byerytheroid differentiation of human leukemia cell lines is inhibited byBct-XL, Blood 87:3837, 1996, using 26 cycles of PCR. The 258 base pairBAX amplicon is amplified using the following primers (Biosynthesis,Lewisville, Tex.) and conditions: BAX-upstream(5′-ACCAAGAAGCTGAGCGAGTGTC-TC-3′) (SEQ ID NO:1), BAXdownstream(5′-CAATGTCCAGCCCATGATGG-3′) (SEQ ID NO:2), cDNA denaturation for oneminute at 94° C., annealing for 15 seconds at 60° C., primer extensionfor 15 seconds at 72° C., with a final extension for 5 minutes. Allsamples are loaded on a five percent nondenaturing polyacrylamide geland electrophoresed for two hours at 80V. For determination ofincorporated radionucleotide, gels are dried down and exposed to aphosphoroimaging plate (Molecular Dynamics, Inc) overnight. Plates arethen scanned on a phosphorimager (Molecular Dynamics) and bandintensities (pixels/unit area) for Bcl-2, Bcl-XI, Bcl-Xs, and BAX areanalyzed normalized to Histone 3.3 expression. PCR amplification of theMDRI, MRP, and LRP genes is performed essentially as described byAbbaszadegan M. et al., Analysis of multidrug resistance-associatedprotein (MRP) messenger RNA in normal and malignant hematapoietic cells,Cancer Res 54:4676,1994, and Komarov P. et al., Activation of the LRPOun-resistance-associated protein) gene by short-term exposure of humanleukemia cells to phorbol ester and cytarabine, Oncology Res 10: 1 85,1998, by using the housekeeping genes histone 3.3 (MDRI) or β-actin (MRPand LRP) as internal standards. cDNA synthesized from 8226/DOX6 RNA isused as a positive control for MDRI PCR. For all reactions, optimalcycle numbers are used and are within the exponential range of PCRamplification as determined by previous experiments.

Intracellular drug accumulation assay

Cells, 0.5×10⁶, are adhered to FN-coated 6-well plates for 24 hours, asdescribed previously. Control wells are coated with BSA or are uncoated.RPMI 1640 containing doxorubicin is added to each treatment well for afinal concentration of 10 μmol/L. After one hour at 37° C., cells arewashed three times with cold PBS and analyzed for FL-2 flourescence on aFACScan machine. Ten-thousand events are recorded for each condition,which are preformed in triplicate. Experiments are repeated twice.

Antibodies and phenotypic analysis of cell lines

Cell surface integrin expression is determined using the monoclonalantibodies (MoAbs) P4G9 (DAKO, Carpinteria, Calif.) for CDw49d (α₄)analysis, A1A5 for CD29 (β₁) analysis (T Cell Diagnostics, Woburn,Mass., PID6 (DAKO) for CDw49e.(α₅) analysis, and FIB504 (Pharmingen, SanDiego, Calif.) for β7 analysis. Cells, 1×10⁶, are incubated with primaryantibody or an isotype control, then with FITC-conjugated goatanti-mouse or goat anti-rat secondary antibody. Fluorescence is thenanalyzed by flow cytometry with a FACScan machine, which records 10,000events for each experiment.

Adhesion assays

Cells, 1.5×10⁵, are adhered to FN- or BSA-coated well plates asdescribed previously. After three washes to remove unattached cells,adherent cells are fixed in 70 percent methanol for 10 minutes.Following aspiration, wells are allowed to dry and then are stained with0.02 percent crystal violet/0.2 percent ethanol for an additional 10minutes. After solubilization with 100 μL Sorenson buffer, absorbance at540 nm is read with an automated microliter plate reader. In someexperiments, cells are pre-incubated for 15 minutes with P4G9 or HP2/1(Clonetech, Palo Alto, Calif.) (anti-VLA-4), PI D6 (anti-VLA-5), orisotype antibody controls before application to wells.

Results

FN-adhered Myeloma Cells Show a Decreased Response to Doxorubicin by MTTCytotoxicity Analysis

A short-term MTT-based cytotoxicity assay is used to assess whether ornot engagement of cell surface integrins can contribute to cellsurvival. 8226/S cells are adhered to FN-coated wells for one hour, andunbound cells are removed by aspiration and washed with serum-freemedia. As a control, an approximately equal cell number is added touncoated wells or wells coated with BSA. After 24 hours, doxorubicin ormelphalan is added to each well for one hour, drug-containing media isthen removed and replaced by fresh media. After a 96 hour incubation,cell survival is determined by the ability of viable cells to reduce MTTdye to formazan. IC50 values are derived through linear regression ofthe log-linear dose-response plots for each cell line to each drug.Student's T-test is used to analyze differences in drug response usingdata collected from three different experiments (0.05 significancelevel). 8226/S myeloma cells adhered to FN-coated plates have asignificant survival advantage over those grown on BSA-coated plateswhen exposed to doxorubicin for 1 hour following a 24 hour pre-adhesionperiod [FIG. 1a], (n=3, mean difference=6.9.s.d.=5.2, range=2.4 to 12.6,P<0.05). The mean IC50 value for FN-adhered cells is 1.63 uM dox(s.d.=1.51, range=0.49 uM to 3.34 uM) compared to 0.52 uM for cellsgrown on BSA (n=3, s.d.=0.76, range.=0.085 uM to 1.4 uM). Subtoxicconcentrations of doxorubicin often induced a mitogenic effect inFN-adhered cells (>100 percent survival. FN-adhered cells often showed adecreased response to melphalan [FIG. 1b], however this effect proved tobe inconsistent (n=3, mean difference=1.7, s.d.=0.8, range=1.2-2.6). Themean IC50 value for FN-adhered cells is 48 uM melphalan (n=3, s.d.=26uM, range=18 uM to 65 uM), compared to 30 uM for cells grown on BSA(n=3, s.d.=20 uM, range=15 uM to 53 uM).

Annexin V Flow Cytometry Analysis of Apoptosis

As a second marker for apoptosis not based on cell growth, phycoerythrin(PE)-conjugated Annexin V is used, which recognizes invertedphosphatidylserine on the exterior of the plasma membrane as an earlystage apoptotic marker.

Approximately 0.5×10⁶ 8226/S cells are adhered to FN-coated or uncoated6 well tissue culture plates for 24 hours before being exposed to either1 uM doxorubicin or 50 uM melphalan. After a 24 hour incubation, cellsare collected and the apoptotic fraction determined using Annexin V-PEstaining and flow cytometric analysis. FN-adhered 8226/S cells have alower percentage of apoptotic cells (mean=16.3 percent) compared tonon-adhered controls (mean=40.3 percent) following a 1 hour doxorubicinexposure (P<0.05) [FIG. 2a]. A smaller, but statistically different(P<0.05), effect is seen with FN-adhered cells treated with 50 uMmelphalan (16.53 percent vs. 21.5 percent) [FIG. 2b]. In furtherexperiments, cells are exposed to drug prior to FN-adhesion in anattempt to rescue them from the consequent initiation of apoptosis.Annexin V staining revealed that FN adhesion is unable to rescue myelomacells following initial exposure to doxorubicin or melphalan.

Bcl-2, Bcl-XL, Bcl-XS, and BAX mRNA Levels Are Unchanged in 8226/SFollowing 24 Hour Adhesion to FN

An RNase protection assay is utilized to observe possibletranscriptional changes in these genes, in order to determine whetherexpression of the Bcl-2 family members known to suppress (Bcl-2 andBcl-XL) or promote (BAX and Bcl-XS) apoptosis are altered in FN-adheredcells. Expression levels and ratios of all Bcl-2 family members arefound to be unchanged, and therefore altered RNA levels of theseapoptosis regulating proteins are not likely responsible for protectingFN-adhered myeloma cells from acute cytotoxic drug exposure [FIG. 3]. Toconfirm these results, semi-quantitative RT-PCR for Bcl-2, Bcl-XI,Bcl-Xs, and BAX is performed on RNA collected from three different cellsamples. As in the RNase protection assay, no significant changes in thelevels of these genes are observed in FN-adhered cells.

Intracellular Doxorubicin Accumulation and Expression of Known DrugTransporters Are Not Altered by FN Adhesion

Because active drug transport is a common mechanism of doxorubicinresistance and because in rare instances ECM adhesion can upregulateP-glycoprotein, possible reductions in intracellular drug accumulationin adhered myeloma cells are examined. A flow cytometry-basedintracellular drug accumulation assay reveales that the concentration ofdoxorubicin, which emits at 573 nm after excitation, in FN-adheredmyeloma cells is equal to that seen in non-adhered controls [FIG. 4].Due to the fact that some drug transporters may alter nuclear drugconcentration with minimal effects on total intracellular drug, RT-PCRis used to investigate whether or not three known transport proteins areupregulated by FN adhesion. Two members of the ABC superfamily oftransmembrane gycoproteins, MDR1 (encoding P-glycoprotein) and MRP(encoding the multidrug resistance-associated protein), which are knownto actively extrude drugs such as doxorubicin, are unchanged followingFN adhesion. Expression of LRP (lung resistance-associated protein),which has also been associated with drug resistance, is also unchangedin adhered myeloma cells compared to suspension cells. In addition toruling out altered drug transport as a mechanism of adhesion-based drugresistance, these studies also show that ECM components of the bonemarrow microenvironment environmental probably do not affect theintrinsic expression of these drug transporters in human myeloma cells.

VLA-4 Is Over Expressed in Drug Resistant Variants of the 8226/S MyelomaCell Line

Low level drug resistant variants are selected from the 8226/S drugsensitive human myeloma cell line using step-wise increases in melphalanor doxorubicin over a period of approximately 10 months. The acquiredresistance of the 8226/LR5 (L-phenylalanine mustard resistant) cell lineis based on the overexpression of glutathione and glutathione-associatedenzymes. 8226/DOX6 (doxorubicin resistant) acquired a P-glycoproteinbased mechanism of resistance after chronic drug selection. Both ofthese cell lines are assayed for changes in cell surface integrinexpression. Cell lines are incubated with monoclonal antibodies to theα₄, α₅, β₁ or β₇ integrin subunits, followed by labeling with afluorescein isothiocyanate (FITC)-conjugated secondary antibody. In both8226 cell line variants, acquired resistance to doxorubicin or melphalanis associated with an increase in α₄ surface expression as determined byfluorescence activated cell sorting analysis [FIG. 5, table 1]. Whencompared to levels found in the 8226/S parent line, α₄ subunitexpression is increased 4 fold in both 8226/LR5 and 8226/DOX6 (n3). β₁subunit expression increased 2.5 fold in 8226/LR5 while a more modestincrease of 70 percent is seen in 8226/DOX6 when compared to parent cellline levels. β₇ integrin, the only other integrin subunit known toheterodimerize with α₄, is increased 3.6 fold in 8226/LR5 but remainedunchanged in 8226/DOX6. α₅ expression levels remained relatively low inboth 8226 drug selected cell lines.

TABLE 1 Fluorescence-activated cell sorter (FACS) analysis of integrinsubunits on drug resistant cell lines. Cell line α₄ α₅ β₁ β₇ 8226/S10.41 14.24 8.43 5.72 8226/LR5 46.53* 11.95 44.63* 40.21* 8226/DOX669.00* 7.32 26.21 18.49 Values reported are the mean fluorescenceintensity of representative histograms from three different experiments.*indicates integrin subunit expression is significantly higher than8226/S at the P > 0.05 level (n = 3).

Drug Sensitive Revertant Cell Lines Express α₄ Levels Equal to that of8226/S

When removed from the drug for a period of 20 weeks, 8226/LR5 revertsback to a drug sensitive phenotype with α₄ integrin expressioncomparable to the drug sensitive parent cell line 8226 [FIG. 6a]. Thelevel of α₅ remained low in the revertant cell line as well (data notshown). The same observations are made when 8226/DOX6 are removed frommaintenance drug for 20 weeks [FIG. 6b]. These experiments demonstrate acorrelation between levels of α₄ expression and drug resistance in the8226 myeloma cell line. Acute exposure of 8226/S to a wide range ofconcentrations of doxorubicin or melphalan has no immediate effects(1-48 hours) on cell surface integrin expression, as determined by FACSanalysis (data not shown), suggesting a process of selection for α₄overexpression, rather than drug-induced upregulation of this gene.

Drug resistant 8226 Cell Lines Demonstrate Increased Levels ofα₄-mediated FN Adhesion

Functionality of surface VLA-4 and VLA-5 is investigated using afibronectin adhesion assay with pre-coated microtiter plates. BSA isused to control for non-specific cell adhesion and several monoclonalantibodies are used to inhibit α₅ (P1D6)- and α₄ (P4G9 andHP2/1)-mediated adhesion. Cell lines selected from 8226/S by continuousdrug exposure displayed significant increases in α₄-mediated FN bindingability [FIG. 7] compared to the parent cell line, 8226/S, whichutilized both α₅ and α₄ for FN adherence. This class switch of integrinsfunctioning in adherence may be indicative of an advantage in α₄- vs.α₅-mediated signal transduction during the selection process, althoughfurther study is needed to investigate these possibilities.

Discussion

Integrins and ECM interactions play critical roles in cell survival.During the course of initial or chronic drug exposure, myeloma cellsoverexpressing VLA-4 have a selection advantage over cells expressinglow levels of this protein. Experiments involving the treatment of8226/S with short term (1-48 hours) doses of doxorubicin or melphalanindicate that increases in α₄ expression are not seen immediately,probably ruling out drug-induced transcriptional upregulation as areason for FN receptor overexpression. Drug resistant cells removed fromchronic drug exposure eventually lose their high α₄ expression levelsalong with their resistant phenotype, implicating selection pressure asa prerequisite for α₄ upregulation. This correlation between integrinexpression and resistance levels is seen in both doxorubicin- andmelphalan-selected variants of 8226/S.

Cells in direct contact with immobilized FN are less sensitive to acutedoxorubicin treatment. A decreased response to melphalan in FN-adheredcells is not consistently observed during acute exposure using MTTcytotoxicity analysis (possibly as a result of assay insensitivity),however, significant increases in cell survival are detected usingAnnexin V. Although the level of cytoprotection may be small followingan acute survival assay, these differences may be sufficient to giverise to a large phenotypically distinct population over the course ofchronic drug exposure in vitro or in.

Soluble FN may have the capacity to induce β₁ signaling without adhesionwithin myeloma cell cultures. The overexpression and utilization of α₄for FN adhesion by drug resistant myeloma cell lines may indicate aconsequent increase in the number of intercellular interactions (throughFN binding, VCAM-1 binding, or homotypic α₄ binding by tumor cellsexposed to DNA damaging agents, or the increased binding of soluble FNfrom serum). Many cultured myeloma cell lines, including 8226, produce arelatively high amount of cell surface FN compared to normal plasmacells, an observation also seen clinically in patient specimens. DNAdamaging agents such as doxorubicin or melphalan may also induceincreased production of ligand, as is shown with human mesangial cellcultures and FN synthesis. Cells under selection pressure may thenutilize soluble or cell-bound integrin ligands, and subsequentα₄-mediated signalling, as a cytoprotective mechanism.

Some previously established mechanisms of drug resistance areinvestigated as possible causes of the cytoprotective effect of FN.Since drug transporters such as P-glycoprotein have been well documentedmediators of drug resistance in myeloma cells, possible alterations inintracellular drug concentration following adhesion are investigated.FN-adhered cells are found to contain doxorubicin levels equal to thoseseen in non-adhered cells using standard techniques. In some cases,intracellular drug compartmentalization can be altered by P-glycoproteinwithout high levels of drug extrusion, for this reason the expression ofthree drug transporters is analzed by semi-quantitative RT-PCR Theexpression of MDR1, MRP, and LRP are all equal between FN-adhered andnon-adhered cells, probably ruling out induction of active drugtransport as a possible mechanism of cytoprotection in theseexperiments.

Another family of proteins known to effect apoptosis and drug responseis the Bcl-2 family. VLA-5 and VLA-6 upregulate Bcl-2 and protectagainst apoptosis in certain cell types following ligation with FN orlaminin, respectively. RNase protection and RT-PCR assays shows the RNAlevels coding for this protein to be unchanged in FN-adhered cells.Furthermore, expression levels of the anti-apoptotic protein Bcl-XL,which is upregulated in keratinocytes following adhesion, are unchangedfollowing FN adhesion. No changes in the expression of otheranti-apoptotic (Bcl-w and Mcl-1) or pro-apoptotic genes (BAX, Bcl-XS,Bad and Bik) are detected by either of these assays. However, it is alsopossible that integrins affect post-translational modification of theseproteins and their potential participation in CAM-DR.

These investigations provide significant insight on the effects ofadhesion on cell cycle kinetics and how this imparts drug resistance totumor cells. VLA-4-mediated adhesion decreases the proliferation ofhematopoietic progenitor cells and the α₄ over expressing cell lines8226/LR5 and 8226/DOX6 have longer doubling times in culture compared tothe 8226/S parent cell line (LR5=30 hr, DOX6=39 hr, S=27 hr).

Phosphatidylinositol 3 kinase (PI3 kinase)/AKT pathway has a majorinfluence on cellular apoptotic commitment and PI3 kinase activationinhibits apoptosis in haemopoietic cells. Thus, this pathway is a likelymechanism of apoptotic suppression in FN-adhered cells. FN adhesion andVLA-4 ligation is known to initiate the PI3 kinase signaling cascade insome cases, but VLA-4 or VLA-5 have not yet been directly correlatedwith this pathway in myeloma cells. Furthermore, a frequent associationis observed between these integrins and FAK, a known activator of PI3kinase. Through activation by PI3-K lipid products or by directphosphorylation, AKT phosphorylates the Bcl-XL/Bcl-2-associated deathpromoter (BAD), promoting cell survival possibly by dissociating it fromBcl-XL and decreasing the amount of BAX homodimers. The endpoint of thePI3-K/AKT signaling cascade may involve an eventual blockade of ced3/ICEactivity and a subsequent inhibition of tumor cell death.

In summary, integrin-mediated FN adhesion is shown to cause a decreasedresponse to chemotherapeutic drugs as well as a correlation between theexpression of α₄ integrin heterodimers and drug resistance. The termcell adhesion mediated drug resistance, or CAM-DR, describes thisobservation. Two well established causes of drug resistance, active drugtransport and increased expression of Bcl-2 family members, are shownnot to produce these effects. Clinically, elevated FN receptorexpression or function in myeloma cells within the bone marrow may be anindicator of a more aggressive tumor cell which has a survival advantageagainst the cytotoxic effects of anticancer drugs. In vivo alterationsin fibronectin receptor expression or function may have a magnifiedeffect on myeloma cell survival when they are in direct association withstromal cells and ECM components of the bone marrow. The cytoprotectionconferred by fibronectin receptors may be low level, but intrinsic,since most myeloma cells inherently express moderate to high levels ofthese integrins. Small changes in drug sensitivity in vitro are probablyhighly relevant clinically since even a 1 percent surviving tumorfraction can have drastic long term consequences. The CAM-DR mediated byFN adhesion may be sufficient to allow the eventual emergence of drugresistance mechanisms such as upregulation of P-glycoprotein, MRP, andalterations in topoisomerase II, which then become the predominantcytoprotective processes. Therefore, specific integrin subunits and thevarious signal transduction elements they utilize, provide promisingtherapeutic targets. Established antagonists of VLA-4 and VLA-5 integrinfunction may serve as chemosensitizers when administered in conjunctionwith conventional chemotherapeutics, leading to higher levels of drugresponse and improved clinical outcome.

EXAMPLE TWO

Materials and Methods

Cell culture conditions

The K562 cell line is obtained from the American Type Culture Collection(Rockville, Md.). Cells are grown in suspension in RPMI 1640 mediumsupplemented with 5 percent fetal bovine serum, 1 percent (v/v)penicillin (100 units/ml), streptomycin (100 units/ml), and 1 percent(v/v) L-glutamnine (all from Gibco BRL, Grand Island, N.Y.). Cells aremaintained at 37° C. in 5 percent CO₂-95 percent air atmosphere andsubcultured every 6 days.

Materials

All cytotoxic drugs are obtained from Sigma-Aldrich (St. Louis, Mo.) andare dissolved in H₂O except for melphalan (LPAM), which is dissolved inacidified ethanol. BCR/ABL kinase inhibitor AG957 is from Calbiochem(San Diego, Calif.) and is solubilized in DMSO. Exposure toγ-irradiation is carried out in a Mark I model 68A irradiator using a2,200 Ci Cs source. Anti-CDw49d (α₄) mAb P4G9, anti-CDw49e (α₅) mAbP1D6, anti-Bcl-2 mAb clone 124, and FITC-conjugated goat-anti-mouseantibodies are all from DAKO (Carpinteria, Calif.). Anti-CD29 mAb MAR4is from Pharmingen (San Diego, Calif.). β1 blocking Ab P4C10 is fromLife Technologies (Rockville, Md.). To stimulate integrin function insome studies, anti-β1 mAb B3B11 (Chemicon, Temecula, Calif.) is used.Anti-Mcl-1 polyclonal Ab, anti-Bax polyclonal Ab, and anti-Bcl-Xpolyclonal Ab are from Santa Cruz Biotechnology (Santa Cruz. Calif.).Anti-phosphotyrosine mAb (4G10) is from Upstate Biotechnology (LakePlacid, N.Y.). Anti-β-actin mAb is from Sigma. HRP-conjugatedgoat-anti-mouse and goat-anti-rabbit antibodies are from JacksonImmunoresearch (West Grove, Pa.).

MTT cytotoxicity assays

Plastic 96-well Immunosorp plates (Nunc, Denmark) are coated with 50 μl(40 μg/ml) FN (Life Technologies) or BSA overnight. Cells are washedonce and resuspended in serum-free RPMI 1640, then 2×10⁴ (K562) cellsare added to each FN coated well. 8×10³ (K562) cells are added to BSAcoated wells. Cells are incubated for 2 hours at 37° C., washed withserum free media, and put back into serum-containing media Following 24hours in a tissue culture incubator, drug or vehicle control is added toeach well for 90 minutes, media is removed and replaced by drug-freemedia for 96 hours. In some experiments, the drug is allowed to remainin each well for 96 hours. 50 μl3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolylblue (MTT dye) (Sigma) is added to each well for 4 hours, plates arecentrifuged and each well aspirated. Dye is solubilized with 100 μl DMSOand plates are read at 540 nm on an automated microtiter plate reader. Ablank well containing only medium and drug is also run as a control inall experiments. IC50 values are calculated by linear regressionanalysis of percent survival vs. drug concentration.

Annexin V apoptosis analysis

For each replicate, 1×10⁶ cells are attached to 35 mm dishes (Nunc)coated overnight with 1 ml of 40 μg/ml FN. In some experiments, cellsare pre-incubated with β1 stimulating mAb B3B11 for 15 minutes at 37°,then are incubated with α4 or α5 blocking antibody for an additional 15minutes prior to attachment. After two hours, non-adhered cells areremoved from plates with serum-free media washes. Cells are exposed todrug for 90 minutes (plus a 24 hour drug-free incubation period) or for24 hours continuously. As a control, 0.4×10⁶ cells are drug treated insuspension and then plated on uncoated 35 mm dishes. Cells are thencollected using gentle agitation, washed, and are incubated withFITC-conjugated Annexin V (BioVision, Palo Alto, Ca). 5000-10000 eventsare analyzed on a FACScan machine (Becton-Dickinson, San Jose, Calif.)and percent apoptosis is determined using CellQuest software(Becton-Dickinson).

Integrin expression analysis

Cells are washed once with cold PBS, incubated with primary antibody oran isotype control for 30 minutes on ice, washed two times with PBS,then incubated with FITC-conjugated goat anti-mouse secondary antibodyfor 30 minutes. Following two washes with PBS, FL-1 fluorescence isanalyzed by a FACScan machine, which recorded 10,000 events for eachanalysis.

Adhesion assays

Cellular adhesion to FN is determined. In some experiments, cells arepre-incubated for 15 minutes with P4G9, P1D6, B3B11, or isotype antibodycontrols prior to application to wells.

Western blotting

Approximately 5×10⁶ cells are centrifuged, resuspended in serum-freeRPMI, and adhered to 60 mm FN-coated plates (3 ml of 40 μg/ml FN isused). Control cells are kept in serum-free media in conical tubes(equal density) for a period of time equal to the adhesion time.Non-adherent cells are gently washed 2-3 times with serum-free media andcells are placed back into media containing 5 percent FBS. Cells arelysed in ice cold modified RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mMNaCl, 1 percent NP-40, 0.5 percent sodium deoxycholate, 0.1 percent SDS)containing fresh protease and phosphatase inhibitors (2 μg/ml aprotinin,2 μg/ml leupeptin, 2 mM PMSF, 20 mM NaF, 1 mM sodium vanadate, 10 mMsodium pyrophosphate, 20 mM β-glycerophosphate). Lysates are cleared bycentrifugation, and quantitated using the BCA protein assay (Pierce,Rockford, Ill.). Equivalent amounts of protein are combined with 6×SDSLaemelli loading dye, boiled for 3 minutes, then separated by 10 percentSDS-PAGE. Proteins are transferred to PVDF membrane (Bio-Rad, Richmond,Calif.) overnight, after which membranes are blocked in PBS containing 5percent dry milk and 0.01 percent tween-20 (Bcl-2 family and β-actinimmunoblotting) or PBS containing 3 percent milk for 2 hours(phosphotyrosine immunoblotting). Membranes are incubated with primaryantibodies and washed as per manufacturer's protocol, then incubatedwith horse radish peroxidase (HRP)-conjugated goat-anti-mouse orgoat-anti-rabbit secondary antibodies for 1 hour. Proteins arevisualized using Lumi-Light substrate (Boehringer Mannheim,Indianapolis, Ind.) and X-ray film (Kodak). To assay for equal proteinloading, membranes are stripped for 30 minutes at 50° C. in strippingbuffer (62.5 nM Tris-HCl pH 6.8, 2 percent SDS, 100 mM2-mercaptoethanol), re-blocked, and probed with anti-β-actin mAb.

RNase protection assay

RNase protection assays are performed. Bcl-2 family specific probes aresynthesized using a template set from Riboquant (San Diego, Calif.).Expression of mRNA corresponding to Bcl-2, Bcl-XI, Bcl-Xs, BAX, Bik,Bad, Bcl-w, Bak, Mcl-1 and Bfl1 is quantitated by normalizing to GAPDHand L32 expression.

α4 vector construction and transfection

The full-length cDNA encoding the human α4 integrin subunit (accession#X16983) is obtained from ATCC in a Bluescript plasmid. The 3.87 KBalpha4 insert is cut out of the plasmid, run on a 1 percent low meltingpoint agarose gel, stained with ethidium bromide, and cut out andpurified using a gel purification kit (Qiagen, Valencia, Calif.). Theinsert is then treated with alkaline phosphatase (Boehringer Mannheim)and ligated into the multiple cloning site of pcDNA3.1(+) (Invitrogen,San Diego, Calif.) using T4 DNA polymerase (Invitrogen). Bacteria aretransformed and colonies are selected for mini-prep analysis. Vectorsare checked for correct orientation of insert using restriction digestsand inserts are sequenced by the molecular biology core (Moffitt CancerCenter). K562 cells are split one day prior to transfection. 2×10⁶ cellsare washed once with PBS and then seeded into a 60 mm dish (Nunc) in 4ml of RPMI 1640/5 percentFBS/1 percent Pen/Strep. 5 μg of vector DNA(pcDNA3.1 or pcDNA3.1/alpha4) dissolved in TE buffer (pH 7.4) iscombined with serum-free media to make a total volume of 150 μl. 20 mlof Superfect reagent (Qiagen) is then added and the mixture is vortexedfor 10 seconds. Following a 10 minute incubation at room temperature, 1ml of media containing serum and antibiotics is added to the mixturewhich is then added dropwise to 60 mm dishes with cells. 48 hours aftertransfection, cells are seeded into T75 flasks (Falcon) and stabletransfectants are selected by adding 1000 μg/ml G418 (Genetecin, LifeTechnologies) to the culture medium for approximately four weeks. α4expression is analyzed by flow cytometry and to select for a morehomogenous population, pcDNA3.1/α4 and pcDNA3.1 transfected cells aresubcloned using a limiting dilution technique. All clones fromK562/pcDNA3.1 are negative for α4 expression by flow cytometery and 3clones from the K562/α4 population are positive. These three clones fromeach line are mixed to create the cell lines K562/pcDNA3.1 (empty vectortransfected) and K562/VLA-4 (α4 transfected).

RT-PCR for a4 Integrin subunit expression

Total cellular RNA is collected from 3×10⁶ cells using TRIzol reagent(Gibco). RNA is quantitated on a spectophotometer at 260 nm. cDNA issynthesized in a 20 ul reverse transcription reaction containing 100 ngRNA, 1×PCR Buffer (10 mM Tris, pH 8.3-50 mMKCL-1.5 mM MgCl₂), 1 mMconcentrations each of dATP, dGTP, dCTP, and dTTP; 100 pmol randomhexamers, 20 units RNAse inhibitor, and 6 units avian megalovirusreverse transcriptase (Boeringer-Mannheim). The reaction is prepared onice then incubated at 42° C. for 1 hour, 99° C. for 10 minutes, andquick chilled to 4°. The following primers are synthesized (Gibco/BRL)and used for α4-specific PCR reactions: 5′-ATGGCTCCCAATGTTAGTGTGG-3′(upstream) (SEQ ID NO:3) and 5′-CACTGGCTTCTTTTCCACTTTCC-3′ (downstream)(SEQ ID NO:4). The 292 base pair α4 product is amplified using 20 μl ofPCR reaction mixture (1×PCR buffer, 6.25 pmol of α4 specific amplimers),0.5 units Taq polymerase (Boehringer-Mannheim), and 5 μl of cDNA.Samples are subjected to incubation at 94° C. for 1 minute and then 34cycles of 94° C. (15 sec), 72° C. (15 sec), and a final extension at 72°C. for 1 minute in a thermal cycler (Perkin-Elmer Cetus). Histone 3.3 isamplified and used as a control for RNA integrity and quantity. Samplesare loaded on a 3 percent agarose gel, electrophoresed one hour at 80V,and visualized using EtBr staining. Restriction digests are used toconfirm identity of products.

Results

K562 Cells Are Resistant to Drug-induced Apoptosis when Adhered to FN

The MTT assay is used to detect differences in the drug sensitivity ofFN adhered vs. suspension-cultured cells following a 90 minutes or 96hours (continuous) exposure to either mitoxantrone or melphalan(L-phenylalanine mustard or LPAM). After a 96 hour period in a cellincubator, cell survival is determined by the ability of viable cells toreduce MTT dye to formazan. IC50 values are derived through linearregression of the log-linear dose-response plots for each cell line toeach drug. Student's T-test is used to analyze differences in drugresponse using data collected from at least three different experiments.FIGS. 8A and 8B depict representative continuous exposure experimentsusing mitoxantrone and LPAM, respectively.

Table 2 summarizes IC50 values, standard deviations, and folddifferences. FN adhered K562 cells exposed to the topoisomerase IIinhibitor mitoxantrone for 90 minutes are approximately 3.7 foldresistant when compared to suspension treated cells (p<0.05; mean IC50suspension=2.06×10⁻⁷M, s.d.=1.62, n=4, range=1.5×10⁻⁸M to 4.04×10⁻⁷M;mean IC50 FN=7.67×10⁻⁷M, s.d.=4.49, n=4, range=5.2×10⁻⁷ M to1.44×10⁻⁶M). Under continuous exposure, FN adhered cells are 6.8 foldmore resistant to mitoxantrone compared to suspension grown cells(p<0.05; mean IC50 suspension=4.79×10⁻⁸M, s.d.=1.64, n=3,range=3.78×10⁻⁸M to 6.68×10⁻⁸M; mean IC50 FN=3.27×10⁻⁷M, s.d.=3.00, n=5,range=1.62×10⁻⁸M to 6.69×10⁻⁷M). FN adhered cells are approximately 6.5fold more resistant to LPAM treatment (90 minutes) compared tosuspension grown cells (p<0.05; mean IC50 suspension=2.93, s.d.=70, n=5,range=1.48×10⁻⁵M to 5.14×10⁻⁵M; mean IC50 FN=1.90×10⁻⁴M, s.d.=1.09, n=5,range=5.85×10⁻⁵M to 3.4×10⁻⁴M). Following a continuous exposure to LPAM,FN adhered cells are 2.25 fold more resistant than suspension cells(p<0.10; mean IC50 suspension=2.55×10⁻⁵M, s.d.=1.42, n=5,range=1.06×10⁻⁵M to 4.32×10⁻⁵M; mean IC50 FN=5.73×10⁻⁵M, s.d.=3.43, n=5,range=2.32×10⁻⁵M to 1.04×10⁻⁴M).

TABLE 2 LPAM- LPAM- Mitoxantrone- Mitoxantrone- 90 min. continuous 90min. continuous exposure exposure exposure exposure Suspension 2.93 ×2.55 × 2.06 × 10⁻⁷M 4.79 × 10⁻⁸M mean IC50 10⁻³M 10⁻⁵M (+/−1.62)(+/−1.64) (+/−1.70) (+/−1.42) Fibronectin 1.90 × 5.73 × 7.67 × 10⁻⁷M3.27 × 10⁻⁷M mean IC50 10⁻⁴M 10⁻⁵M (+/−4.49) (+/−3.00) (+/−1.09)(+/−3.43) Fold resistance 6.48 2.25 3.72 6.83

As a second method for determining cell survival, an Annexin V flowcytometric based assay is employed. Cells are seeded onto dishes coatedwith FN, poly-L-lysine (PLL), or BSA (or are kept in suspension) for 2hours in serum-free media, exposed to drug for 90 minutes, washed, andincubated drug-free for an additional 24 hours. In some experiments,cells are exposed to drug for 24 hours (continuous exposure). Thepercent of apoptotic cells is then determined by staining with AnnexinV, which recognizes inverted phosphatidyl serine on the external surfaceof the cell as an early apoptotic marker.

FIG. 9 demonstrates that FN adhesion confers significant protection fromapoptosis induced by LPAM exposure. In these experiments, BSA is used asa control for non-specific protein interactions and PLL is used as acontrol for non-specific cell adhesion. Neither of these substratesconfers a survival advantage to cells when compared to those treated insuspension. It is interesting to note that the addition of soluble FN toconditioned serum-free culture media does not cause a consistentsuppression of drug-induced apoptosis, suggesting the CAM-DR phenotyperequires both ligand occupancy of the FN receptor and integrin-specificcell adhesion. Table 3 summarizes drug and radiation responses of K562cells adhered to FN vs. grown in suspension. Significant cytoprotectionis primarily observed for DNA damaging agents (FN-mediated protectionagainst the microtubule inhibitor vincristine is not significant). FNadhesion also afforded a higher degree of protection following acute (90minutes) exposure to drug when compared to continuous exposure.Vincristine and Ara-C are only used in continuous exposure experimentsdue to the inherent insensitivity of K562 cells to these compounds.

TABLE 3 Suspension: FN: % Control Treatment mean death mean death deathLPAM 100_(μ)M 37.5 ± 24.0 11.0 ± 8.3 29.3* 1 hr LPAM 100_(μ)M 39.8 ± 3.223.3 ± 6.4 58.5* cont. exp. Mitox 50_(μ)M 46.3 ± 8.4 27.0 ± 9.4 58.3* 1hr Mitox 10_(μ)M 42.8 ± 8.4 29.7 ± 6.2 69.4* cont. exp. VCR 10_(μ)M 17.8± 6.4 12.5 ± 2.3 70.2 cont. exp. Ara-C 100_(μ)M 11.3 ± 5.5  6.2 ± 1.654.9* cont. exp. XRT 5000 32.3 ± 5.5 26.4 ± 3.6 81.7* rads

Generation and Characterization of Stable VLA-4 Transfectants

Although the K562 cell line expresses only the VLA-5 FN receptor, CMLcells from patients also express the VLA-4. Other cell types also showthe anti-apoptotic potential of VLA-4. For these reasons, an α4 positiveK562 cell line is created for use in subsequent experiments. A vectorcontaining the full coding sequence for the human α4 integrin subunit iscreated and transfected into the K562 cell line. As a control, anotherpopulation of K562 cells is also transfected with an empty vectorconstruct (pcDNA3.1). Following a 4 week culture period with Geneticin(G418) to select for stable positive transfectants, cell populations areanalyzed by flow cytometry for cell surface integrin expression. Alimiting dilution assay is then used to subclone both high α4 expressingand α4 negative cell lines. Three independent positive and negativeclones are grown to confluency and pooled to make the K562/VLA-4 andK562/pcDNA3 cell lines, respectively.

FIG. 10A shows cell surface expression of both the β1 and α5 integrinsubunits on the surface of both cell lines but the presence of α4 onlyon the K562/VLA-4 cell line. RT-PCR using α4 specific primers shows thatthe α4 message is only detectable in the K562/VLA-4 cell line (FIG.10B).

The ability of transfected cell lines to adhere to FN through variousintegrin subunits is determined using a colorimetric cell adhesion assayand function-blocking antibodies to ⊕1, α4 and α5. Cells arepre-incubated with integrin-specific antibodies or control antibodies(non-specific mouse IgG) and then seeded onto FN coated 96 well platesfor 90 minutes. Although a high level of VLA-4 is expressed on thesurface of K562/VLA-4, these cells are incapable of adhering to FNthrough α4, indicating that VLA-4 is in an inactive conformation.Addition of a β1-stimulating to mAb (clone B3B11) is necessary to induceVLA-4-mediated FN adhesion in K562/VLA-4 but not K562/pcDNA3.1 (FIG.11). Incubation with α4 blocking antibody does not significantly reduceFN adhesion in either cell line, indicating that this subunit is eithernot expressed (in the case of K562/pcDNA3.1) or is not required foradhesion in the presence of the overabundance of VLA-5 (K562/VLA-4).Incubation with α5 blocking antibody blocked almost all adhesion ofK562/pcDNA3.1 to FN but only slightly reduced the adhesive capacity ofK562/VLA-4. These experimentsshow that K562/pcDNA3.1 adheres to FNsolely through VLA-5 (as does the K562 parent cell line), whileK562/VLA-4 utilizes both VLA-4 and VLA-5 for FN adhesion.

Contribution of α4 and α5 to CAM-DR in K562

K562/pcDNA3.1 and K562/VLA-4 cells are incubated with mAb B3B11 andanti-α4 or anti-α5 blocking Ab prior to adhesion to FN coated 35 mmdishes to determine whether or not α4 can contribute to CAM-DR inaddition to α5 in CML cells. Following a two hour adhesion period,plates are washed with serum-free media (suspension control cells alsowashed), and media containing 100 μM LPAM or Acid-OH vehicle control isthen added to each sample for 90 minutes and cells are then incubatedfor an additional 24 hours. Apoptosis is analyzed using Annexin Vstaining and flow cytometry. As can he seen in FIG. 12, K562/VLA-4 cellsadhering through both α4 and α5 integrins demonstrate a level ofcytoprotection (in comparison to drug treated suspension cells)equivalent to K562/pcDNA3.1 cells adhered through α5 alone (30.0±1.5percent vs. 29.9±5.3 percent). When incubated with α4 blocking antibodyprior to FN adhesion, both cell lines still demonstrated significantprotection against melphalan-induced apoptosis (28.4±5.0 percent vs.29.6±2.3 percent). However, when incubated with a blocking antibodyprior to adhesion, the K562/pcDNA3.1 cells became sensitized to drugwhereas the K562/VLA-4 cells remained significantly resistant (0±11.1percent vs: 20.6±1.9 percent). It can be concluded that VLA-5 and VLA-4are capable of inducing cytoprotection in K562 cells through similarmechanisms since the effects of both integrins are not additive comparedto either one alone. CD34+CML cells are known to express both of theseintegrins in vivo and it can be inferred that if either are functional,the CAM-DR phenotype may be initiated through their common β1 integrinchain.

FN Adhered K562 Cells Are Resistant to Apoptosis Induced by BCA/ABLKinase Inhibition

The tyrphostin AG957 is used as a specific inhibitor of thep210^(bcr-abl) kinase to investigate the effects of integrin-mediated FNadhesion on cell death following inhibition of the BCR/ABL cell survivalpathway. The MTT cytotoxicity assay is again utilized to assesssensitivity to this compound in FN adhered vs. suspension-grown K562cells. FIG. 13 is a representative curve showing the high degree ofcytoprotection afforded by FN adhesion following exposure to AG957. Fromthree independent experiments, the mean IC50 value for suspension growncells is 3.60×10⁻⁶M (s.d.=2.74, range=7.90×10⁻⁷M to 6.27×10⁻⁶ M)compared to 12.95×10⁻⁶M (s.d.=3.53, range=9.56×10⁻⁶M to 16.60×10⁻⁶M) forFN adhered cells. These values are significantly different at the p<0.05level (by Student's t-test) and the fold resistance of FN adhered cellsover suspension cells is 3.6.

BCR/ABL-associated Tyrosine Kinase Activity Is Not Reconstituted byIntegrin-Mediated Signaling

The BCR/ABL protein is responsible for the constitutive phosphorylationof many kinases associated with integrin-mediated signaling K562 cellsadhered to FN have a higher survival rate than suspension grown cellsfollowing AG957 treatment This leads to the hypothesis that VLA-5 iscausing cell survival by substituting for BCR/ABL and inducing theactivation of proteins such as p125FAK, p130Cas, PI-3 kinase, paxillin,and c-Abl. A temporal analysis of phosphotyrosine containing proteins bywestern blotting demonstrates that there is no further increase in theactivity of kinases already active due to BCR/ABL, with the exception fan unknown 80 kDa protein (FIG. 14). Following a 4 hour treatment with20 μM AG957, BCR/ABL associated tyrosine phosphorylation is completelyabrogated in suspension cells (FIG. 15). In AG957 treated cells, FNadhesion does not reconstitute the phosphotyrosine pattern seen inuntreated cells, however the 80 kDa band persists, albeit to a lesserextent. These results indicate that the mechanism of CAM-DR in K562 isindependent of the BCR/ABL survival pathway, or at least the portioninitiated by tyrosine kinase activation.

FN Adhered K562 Cells Maintain the CAM-DR Phenotype During AG957Exposure

With the exception of an unknown 80 kDa protein, FN adhered cells do notinduce noticeable tyrosine phosphorylation following AG957 treatment(FIG. 15). It is unclear whether or not FN adhered cells would still beresistant to drug-induced apoptosis following AG957 exposure. Cells areadhered to FN or kept in suspension for 3 hours, then exposed to AG957(10 or 20 μM) or DMSO vehicle control for 1 hour, and then treated with100 μM LPAM or vehicle control for 90 minutes. Apoptosis is analyzed byAnnexin V staining 24 hours later. As can be seen in FIG. 16, the CAM-DRphenotype is maintained even when FN adhesion is followed by AG957treatment. Integrin activation is sufficient to protect K562 cells fromthe BCR/ABL inhibitor alone (Acid-OH LPAM controls) and in combinationwith the alkylating agent. These results further support the findingthat integrin-induced cytoprotection is mediated through signalingevents independent of the tyrosine kinases activated by BCR/ABL.

Discussion

Myeloma cells adhered to FN are resistant to drug-induced apoptosis.Similarly, small cell lung cancer cells adhered to FN, collagen, andlaminin are resistant to cytotoxic agents. Tumor cell-ECM interactionsare also critical determinants in the survival of chronic myelogenousleukemia cells. K562 cells adhered to FN are significantly resistant toapoptosis induced by a number of DNA damaging agents including LPAM,mitoxantrone, and γ-irradiation. Non-specific, charge-based adhesion topoly-L-lysine coated surfaces did not result in decreased drug response.In addition, soluble FN added to culture medium did not provide asurvival advantage to drug treated cells. These results indicate arequirement for both receptor occupancy by FN as well as physicalattachment by the cell in order for cell adhesion mediated drugresistance (CAM-DR) to occur. It remains to be determined whichcytoskeletal elements may be required for a cytoprotective signal to beinitiated when cells become adherent.

When compared to drug sensitive myeloma cells, drug resistant cellsoverexpress the integrin VLA-4 on their cell surface, indicating thisintegrin heterodimer may be critical for the emergence of a drugresistant cell population. VLA-4 may also have importance with regard tothe survival of B and T cells. CAM-DR is mediated by VLA-5 in K562 cellswhich are VLA-4 negative). It would thus be useful to determine if VLA-4could be cytoprotective in these cells and whether a combination of FNreceptor subtypes could lead to enhanced cell survival above and beyondthat mediated by either receptor alone. Since CML cells isolated frompatients are known to express both VLA-4 and VLA-5, this informationcould prove to be useful for the future application of pharmacologicalinhibitors against α4, α5, or β1 integrin subunits in this disease.

When adhered to FN, K562/pcDNA3.1 cells (expressing only VLA-5) andK562/VLA-4 cells (expressing both VLA-5 and VLA-4) are found to havecomparable responses when treated with LPAM. Blocking antibodyexperiments also demonstrate that VLA-4 by itself could also becytoprotective in K562 cells. It can therefore be concluded that each ofthese integrins share similar signaling capabilities likely based ontheir common β1 subunit.

The BCR/ABL kinase is believed to be critical to the survival of CMLcells through its effects on a number of signal transduction pathways.Inhibitors of this fusion protein show promise as apoptosis-inducingdrugs in CML cells. BCR/ABL inhibition by relatively high doses of thetyrphostin AG957 can have negative effects on FN adhesion. At theconcentrations of inhibitor used in cytotoxicity assays, the adhesion ofK562 cells to FN is not affected by more than 20-40 percent.

In addition to the cytotoxic agents examined, K562 CML cells are foundto be resistant to the BCR/ABL inhibitor AG957 when adhered to FN. Theseobservations show that integrins, in at least the K562 cell line, maysignal through tyrosine kinases similar to those activated by BCR/ABL,effectively reconstituting the survival pathway inhibited by AG957. Insome instances, when activated through FN adhesion, integrins are knownto mimic many of the aspects of BCR/ABL signaling through therecruitment of c-Abl. Proteins such as p125FAK, p130Cas, paxillin, andPI3-kinase are activated (through tyrosine phosphorylation) by bothBCR/ABL and β1 integrins independently. Therefore, FN adhesion allowK562 cells to survive BCR/ABL inhibition by substituting as theinitiator of this survival pathway. However, it is discovered that thetyrosine phosphorylation pattern induced by the BCR/ABL kinase iscompletely abrogated by AG957 in both suspension and FN adhered cells(FIG. 8), indicating that integrin ligation is providing a survivalsignal independent of this group of signaling proteins. A protein bandof approximately 80 kDa is activated by FN adhesion and persisted, to alesser degree, in AG957 treated cells. Note that this protein isaffected as well by AG957, possibly indicating a degree ofnon-specificity of this compound. Although these experiments rule outthe integrin-induced activation of focal adhesion proteins such as FAK(125 kDa), p130Cas (125 and 115 kDa), paxillin (65 kDa) and c-Abl (145kDa), other β1 integrin-activated proteins that are not detected byphosphotyrosine analysis, such as serine/threonine phosphorylatedproteins, may contribute to the CAM-DR phenotype.

γ-irradiation and drugs such as melphalan, mitoxantrone, and AG957induce apoptosis through different means. The ability of β1 integrins toprotect cells from apoptosis induced by such a wide range offunctionally distinct agents suggests a block in the initiation orexecution of apoptosis at a point of general convergence. The Bcl-2family of proteins fits the profile of general regulators of apoptoticcommitment and these proteins are affected by integrin activation. AnRNase protection assay demonstrates no consistent changes in the levelsof RNA pertaining to any one of ten Bcl-2 family member genes in FNadhered K562 cells. In addition, the protein levels of Bcl-2, Bcl-Xl,Bax, and Mcl-1 are not altered by FN adhesion in K562 cells. This isconsistent with findings reported for the RPMI 8226 myeloma cell line.

The ECM protein FN has been found to play a key role in the survival ofa number of hematopoietic cell lines under conditions of cytotoxicstress. CML cells remaining in contact with the bone marrow may form thebasis of a tumor cell population that is resistant to theapoptosis-inducing effects of chemotherapy. The cellular changes inducedby the VLA-4 and VLA-5 integrin molecules seem to be mediated by β1since each of these receptors are capable of providing cytoprotection,but not additively. In vitro, the CAM-DR phenotype also allows cells tosurvive the apoptosis-inducing actions of AG957, a specific inhibitor ofthe BCR/ABL kinase. AG957 is able to disrupt the tyrosinephosphorylation of all proteins activated by the BCR/ABL kinase in K562cells at relatively low concentrations, leading to cell death. Integrinactivation, despite providing protection against BCR/ABL inhibition, didnot result in the phosphorylation of any of these proteins. Finally, itis determined that FN adhered K562 cells remain resistant to melphalanfollowing AG957 exposure, a further indication that the mechanism ofCAM-DR is independent of proteins within the BCR/ABL cell survivalpathway. In conclusion, β1 integrins implicated in the induction of drugresistance of K562 CML cells, among other hematopoietic cell lines.Small molecule antagonists against the β1 integrin subunit thereforewill prove beneficial as chemosensitizers in the treatment of cancer.

In order to further illustrate the present invention and the advantagesthereof, the following additional examples are given, it beingunderstood that same are intended only as illustrative and in no waylimitative.

EXAMPLE THREE

A pre-established microtiter adhesion assay is used to determine ifpeptides such as RZ-3 are effective at preventing adhesion of myelomacells to FN. The peptide RZ-3 contains all D-amino acids, and has thesequence:

kviywkag (SEQ ID NO:6)  (RZ-3)

in which the conventional one-letter code is used and lower casedesignates a D-amino acid, and the sequence is written from theN-terminus to the C-terminus.

Peptide variants of varying composition are examined for inhibitingtumor cell adhesion to FN and other bone marrow matrices. VLA-4 andVLA-5 are shown to play a major role in maintaining hematopoietic cellswithin the bone marrow and for keeping myeloma cells in an environmentwhich promotes tumor growth and/or blocks apoptosis.

EXAMPLE FOUR

To determine if peptides such as RZ-3 are able to sensitize pre-adheredmyeloma cells to cytotoxic drugs in-vitro, cells are exposed to variousconcentrations of peptides both before and after adhesion to FN andexposure to cytotoxic drugs. Changes in the percentages of cellsundergoing apoptosis are examined by using MTT based cytotoxicity assaysand Annexin V flow cytometric analysis.

EXAMPLE FIVE

To determine if peptides are able to prevent or reverse FN induced G1cell to cycle arrest cells allowed to adhere for 24 to 48 hours beforethe addition of the peptide. Cell cycle arrest is detected usingpropidium iodide and BudR cell cycle analysis as measured by flowcytometry. Presumably malignant cells are already attached to theextracellular matrix before treatment with chemotherapy. Thus, the mosteffective agents will reverse, as well as prevent, cell adhesion to FN.

EXAMPLE SIX

To investigate the effect of peptides in disrupting β1 mediated signaltransduction in myeloma cells the effects of peptides on Bcl-xlexpression in myeloma cells is measured using western blot and RTIPCRanalysis. Results indicate that FN mediated adhesion is capable ofupregulating Bcl-xl expression in myeloma cells which could blockapoptosis.

CONCLUSION

The present invention addresses the problem of chemotherapy andradiation induced drug resistance and describes methods for enhancingthe efficacy of both cytotoxic drugs and radiation in the treatment ofcancer.

The tumor cell microenvironment influences the way a tumor cell behavesand responds to cytotoxic drugs. The microenvironment can enhance tumorcell survival and prevent drug-induced apoptosis. This interactionbetween tumor cell and environment explains how some tumor cells surviveinitial drug exposure and eventually express classical mechanisms ofdrug resistance. Two different forms of tumor cell-environmentinteraction are disclosed. In the first form, soluble chemicalmodulators, such as cytokines, are secreted by non-tumor, stromal cells.These soluble modulators interact with tumor cell surface receptors,which in turn activate signal transduction pathways that enhance tumorcell survival and prevent apoptosis. Interleukin-6 appears to be aclassical example of a soluble modulator secreted by the tumormicroenvironment capable of increasing tumor cell survival andpreventing apoptosis. Approaches to block signal transduction pathwaysmediated by IL-6, or other soluble modulators of apoptosis, can enhancecytotoxic drug activity.

In the second form of tumor cell-environment interaction, direct tumorcell contact with other cells or the ECM is associated with drugresistance. This form of drug resistance has been given the term celladhesion mediated drug resistance (CAM-DR). To date, β1 integrinadhesion and activation mediates the most well characterized CAM-DRphenotype. Other cell adhesion molecules are also likely to play a rolein the CAM-DR phenotype. Interrupting this cell adhesion, or the signaltransduction pathways associated with it, represents a new drug targetfor the treatment of cancer.

These two forms of tumor cell-environment interaction are illustrated byFIG. 28.

In summary, it is herein disclosed that cancer cell interaction with theextracellular matrix, including fibronectin and collagen, prevents celldeath (apoptosis) induced by cytotoxic drugs and radiation, a phenomenondescribed as “Cell Adhesion Mediated Drug Resistance” (CAM-DR). Further,it is disclosed that integrin-mediated adhesion, including α₄β₁ and α₅β₁for fibronectin and α₂β₁ for collagen, prevents both drug and radiationinduced cancer cell death. Therefore, integrin-mediated adhesion confersa survival advantage to cancer cells that have been exposed to drugs orradiation.

It is also shown that specific agents including peptides such as thesynthetic D-amino acid peptide RZ-3 can be utilized to inhibitintegrin-mediated cell adhesion thereby rendering cancer cells moresensitive to chemotherapeutic agents. Thus, integrin-mediated celladhesion confers resistance to chemotherapeutic agents and represents anovel and specific target for the development of therapies that caninterfere with or inhibit CAM-DR. Furthermore, inhibition of CAM-DR hasthe potential to enhance treatment responses by sensitizing cancer cellsto both chemotherapy and radiation therapy.

In particular, the use of peptides (such as RZ-3) for inhibitingadhesion and enhancing the efficacy of chemotherapeutic and/or radiationtreatments in the treatment of cancer is disclosed.

In a preferred embodiment, the peptide is administered prior to theadministration of chemotherapy and/or radiation.

References cited through this application are each herein incorporatedin their respective entireties.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

6 1 24 DNA Artificial Sequence BAX upstream primer 1 accaagaagctgagcgagtg tctc 24 2 20 DNA Artificial Sequence BAX downstream primer 2caatgtccag cccatgatgg 20 3 22 DNA Artificial Sequence upstream primer 3atggctccca atgttagtgt gg 22 4 23 DNA Artificial Sequence downstreamprimer 4 cactggcttc ttttccactt tcc 23 5 9 PRT Artificial Sequenceinhibitor of cell adhesion mediated drug resistance 5 Lys Met Val IleTyr Trp Lys Ala Gly 1 5 6 9 PRT Artificial Sequence RZ-3 6 Lys Met ValIle Tyr Trp Lys Ala Gly 1 5

What is claimed is:
 1. A method for inhibiting cell adhesion mediateddrug resistance in a patient in need thereof, said method comprisingadministering to the patient an effective amount of a peptide thatinhibits cell adhesion mediated drug resistance, wherein the peptidecomprises the amino acid sequence of SEQ ID NO:6, or a pharmaceuticallyacceptable salt or variant thereof, and wherein the variant has at leastone of the following characteristics: the variant contains one or morehydroxyproline or hydroxylysine; the variant contains an organic acid oramide; the variant contains a C-terminal carboxylate; the variant isesterified; the variant is amidated; the variant has a modifiedN-terminal amino group; the variant has a side-chain modification orsubstitution selected from the group consisting of methylation,benzylation, t-butylation, tosylation, and alcoxycarbonylation; thevariant has an N-acetyl group; the variant has a C-terminal amide group;or the variant has an amino acid selected from the group consisting ofpenicillamine, tetramethylene cysteine, pentamethylene cysteine,mercaptopropionic acid, pentamethylene-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, aminoapidic acid, m-aminomethylbenzoic acid, anddiaminopropionic acid.
 2. The method of claim 1, wherein the peptidecomprises the amino acid sequence of SEQ ID NO:6, or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 1, wherein the peptidecomprises the amino acid sequence of SEQ ID NO:6.
 4. A method forenhancing the efficacy of chemotherapy or radiation therapy in a patientin need thereof, said method comprising administering an effectiveamount of a peptide that inhibits cell adhesion mediated drug resistancein the patient; and administering chemotherapy, radiation therapy, orboth, to the patient; whereby the efficacy of the therapy is enhancedand wherein the peptide comprises the amino acid sequence of SEQ ID NO:6, or a pharmaceutically acceptable salt or variant thereof, wherein thevariant has at least one of the following characteristics: the variantcontains one or more hydroxyproline or hydroxylysine; the variantcontains an organic acid or amide; the variant contains a C-terminalcarboxylate; the variant is esterified; the variant is amidated; thevariant has a modified N-terminal amino group; the variant has aside-chain modification or substitution selected from the groupconsisting of methylation, benzylation, t-butylation, tosylation, andalcoxycarbonylation; the variant has an N-acetyl group; the variant hasa C-terminal amide group; or the variant has an amino acid selected fromthe group consisting of penicillamine, tetramethylene cysteine,pentamethylene cysteine, mercaptopropionic acid,pentamethylene-mercaptopropionic acid, 2-mercaptobenzene,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,aminoapidic acid, m-aminomethylbenzoic acid, and diaminopropionic acid.5. The method of claim 4, wherein the peptide comprises the amino acidsequence of SEQ ID NO:6, or a pharmaceutically acceptable salt thereof.6. The method of claim 4, wherein the peptide comprises the amino acidsequence of SEQ ID NO:6.
 7. A method for treating cancer in a patient inneed thereof, said method comprising administering an effective amountof a peptide to the patient to inhibit cell adhesion mediated drugresistance in the patient; and administering chemotherapy, radiationtherapy, or both, to the patient; and wherein the peptide comprises theamino acid sequence of SEQ ID NO: 6, or a pharmaceutically acceptablesalt or variant thereof, wherein the variant has at least one of thefollowing characteristics: the variant contains one or morehydroxyproline or hydroxylysine; the variant contains an organic acid oramide; the variant contains a C-terminal carboxylate; the variant isesterified; the variant is amidated; the variant has a modifiedN-terminal amino group; the variant has a side-chain modification orsubstitution selected from the group consisting of methylation,benzylation, t-butylation, tosylation, and alcoxycarbonylation; thevariant has an N-acetyl group; the variant has a C-terminal amide group;or the variant has an amino acid selected from the group consisting ofpenicillamine, tetramethylene cysteine, pentamethylene cysteine,mercaptopropionic acid, pentamethylene-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, aminoapidic acid, m-aminomethylbenzoic acid, anddiaminopropionic acid.
 8. The method of claim 7, wherein the peptidecomprises the amino acid sequence of SEQ ID NO:6 or a pharmaceuticallyacceptable salt thereof.
 9. The method of claim 7, wherein the peptidecomprises the amino acid sequence of SEQ ID NO:6.
 10. The method ofclaim 7, wherein the cancer is a myeloma.
 11. The method of claim 7,wherein the cancer is multiple myeloma.
 12. An isolated peptidecomprising the amino acid sequence of SEQ ID NO:6, or a pharmaceuticallyacceptable salt or variant thereof, wherein said variant has at leastone of the following characteristics: said variant contains one or morehydroxyproline or hydroxylysine; said variant contains an organic acidor amide; said variant contains a C-terminal carboxylate; said variantis esterified; said variant is amidated; said variant has a modifiedN-terminal amino group; said variant has a side-chain modification orsubstitution selected from the group consisting of methylation,benzylation, t-butylation, tosylation, and alcoxycarbonylation; saidvariant has an N-acetyl group; said variant has a C-terminal amidegroup; or said variant has an amino acid selected from the groupconsisting of penicillamine, tetramethylene cysteine, pentamethylenecysteine, mercaptopropionic acid, pentamethylene-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, aminoapidic acid, m-aminomethylbenzoic acid, anddiaminopropionic acid.
 13. An isolated peptide comprising the amino acidsequence of SEQ ID NO:6, or a pharmaceutically acceptable salt thereof.14. An isolated peptide comprising the amino acid sequence of SEQ IDNO:6.
 15. A pharmaceutical composition comprising an isolated peptideand a pharmaceutically acceptable carrier, wherein said peptidecomprises the amino acid sequence of SEQ ID NO:6, or a pharmaceuticallyacceptable salt or variant thereof, wherein said variant has at leastone of the following characteristics: said variant contains one or morehydroxyproline or hydroxylysine; said variant contains an organic acidor amide; said variant contains a C-terminal carboxylate; said variantis esterified; said variant is amidated; said variant has a modifiedN-terminal amino group; said variant has a side-chain modification orsubstitution selected from the group consisting of methylation,benzylation, t-butylation, tosylation, and alcoxycarbonylation; saidvariant has an N-acetyl group; said variant has a C-terminal amidegroup; or said variant has an amino acid selected from the groupconsisting of penicillamine, tetramethylene cysteine, pentamethylenecysteine, mercaptopropionic acid, pentamethylene-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, aminoapidic acid, m-aminomethylbenzoic acid, anddiaminopropionic acid.
 16. The pharmaceutical composition of claim 15,wherein said peptide comprises the amino acid sequence of SEQ ID NO:6 ora pharmaceutically acceptable salt thereof.
 17. The pharmaceuticalcomposition of claim 15, wherein said peptide comprises the amino acidsequence of SEQ ID NO:6.